Accurate deduction of fetal DNA fraction with shallow-depth sequencing of maternal plasma

Abstract

Embodiments of the present invention provide methods, systems, and apparatus for deducing the fetal DNA fraction in maternal plasma without using paternal or fetal genotypes. Maternal genotype information may be obtained from a maternal-only DNA sample or may be assumed from shallow-depth sequencing of a biological sample having both maternal and fetal DNA molecules. Because sequencing may be at shallow depths, a locus may have only few reads and may fail to exhibit a non-maternal allele even if a non-maternal allele is present. However, normalized parameters that characterize non-maternal alleles sequenced can be used to provide an accurate estimate of the fetal DNA fraction, even if the amount of non-maternal alleles is in error. Methods described herein may not need high-depth sequencing or enrichment of specific regions. As a result, these methods can be integrated into widely used non-invasive prenatal testing and other diagnostics.

What is claimed is: 
     
       1. A method for measuring a fetal DNA fraction in a biological sample of a female pregnant with a fetus, the biological sample including maternal DNA molecules and fetal DNA molecules, the method comprising:
 sequencing a first aliquot of the biological sample to obtain a first plurality of reads from a first plurality of DNA molecules; 
 receiving a first data set from the first plurality of reads; 
 identifying locations of the first plurality of reads in a reference genome; 
 measuring sizes of the DNA molecules corresponding to the first plurality of reads; 
 identifying a first set of loci in the first data set, wherein DNA molecules corresponding to reads located at each of the first set of loci have a first size distribution and have a first size value of the first size distribution exceeding a first size threshold; 
 measuring a first amount of loci in the first set of loci; 
 sequencing a second aliquot of the biological sample to obtain a second plurality of reads from a second plurality of DNA molecules; 
 receiving a second data set from the second plurality of reads; 
 identifying locations of the second plurality of reads in the reference genome; 
 measuring sizes of the DNA molecules corresponding to the second plurality of reads; 
 identifying a second set of loci in the second data set, wherein:
 each one of the second set of loci is one of the first set of loci, and 
 DNA molecules corresponding to reads at each of the second set of loci have a second size distribution and have a second size value of the second size distribution exceeding a second size threshold in an opposite direction than the first size value exceeds the first size threshold; 
 
 measuring a second amount of loci in the second set of loci in the second data set; 
 determining a value of a normalized parameter of the first amount and the second amount; 
 comparing the value to a calibration point that is determined using at least one other sample having a known fetal DNA fraction and a calibration value corresponding to separate measurement of the normalized parameter in the at least one other sample; and 
 computing the fetal DNA fraction based on the comparison. 
 
     
     
       2. The method of  claim 1 , wherein the first size value is larger than the first size threshold and the second size value is smaller than the second size threshold, and wherein the second size threshold is smaller than the first size threshold. 
     
     
       3. The method of  claim 1 , wherein the first size value is smaller than the first size threshold and the second size value is larger than the second size threshold, and wherein the second size threshold is larger than the first size threshold. 
     
     
       4. The method of  claim 1 , wherein a difference between the first size value and the second size value is at least 10 bp. 
     
     
       5. The method of  claim 1 , wherein the first plurality of DNA molecules comprises maternal DNA from a pregnant female, and the second plurality of DNA molecules comprises maternal DNA and fetal DNA from the pregnant female. 
     
     
       6. The method of  claim 5 , further comprising:
 identifying an allele in one or more second reads in the second plurality of reads at a locus in the second set of loci that is not present in one or more first reads in the first plurality of reads at the same locus in the first set of loci; 
 determining one or more first sizes of one or more first DNA molecules corresponding to the one or more first reads; 
 determining one or more second sizes of one or more second DNA molecules corresponding to the one or more second reads exhibiting the allele; 
 comparing the one or more second sizes of DNA molecules exhibiting the allele in the second plurality of reads at the locus with the one or more first sizes of DNA molecules in the first plurality of reads at the locus; and 
 determining whether the allele is a non-maternal allele using the comparison of the sizes of DNA molecules. 
 
     
     
       7. The method of  claim 1 , wherein the first plurality of DNA molecules and the second plurality of DNA molecules are from the biological sample. 
     
     
       8. The method of  claim 1 , wherein:
 the calibration value is determined using a third plurality of reads obtained from the at least one other sample, 
 and the first plurality of reads has a number of reads within 10× of the third plurality of reads. 
 
     
     
       9. The method of  claim 8 , wherein the second plurality of reads has a number of reads within 10× of the third plurality of reads. 
     
     
       10. The method of  claim 1 , further comprising:
 receiving the biological sample; and 
 sequencing the second plurality of DNA molecules in the biological sample to obtain the second plurality of reads. 
 
     
     
       11. The method of  claim 10 , further comprising:
 sequencing the first plurality of DNA molecules in the biological sample to obtain the first plurality of reads. 
 
     
     
       12. The method of  claim 1 , wherein:
 the first plurality of reads provides at or less than 1× coverage of a haploid human genome, and 
 the second plurality of reads provides at or less than 1× coverage of the haploid human genome. 
 
     
     
       13. The method of  claim 1 , wherein:
 the first size value is a median size, a mean size, or a mode of the first size distribution, and 
 the second size value is a median size, a mean size, or a mode of the second size distribution. 
 
     
     
       14. The method of  claim 1 , wherein the first set of loci is chosen from a reference database. 
     
     
       15. The method of  claim 1 , wherein the normalized parameter comprises the second amount divided by the first amount or the second amount divided by the sum of the first amount and the second amount. 
     
     
       16. The method of  claim 1 , wherein:
 the first plurality of reads provides at or less than 1× coverage of a haploid human genome, and 
 the second plurality of reads provides at or less than 1× coverage of the haploid human genome. 
 
     
     
       17. The method of  claim 1 , wherein a difference between the first size value and the second size value is at least 20 bp. 
     
     
       18. The method of  claim 1 , wherein the first plurality of reads includes less than or equal to 50 million reads.